Myosin VIIA is a cellular motor protein which is expressed in a several tissues including the inner ear and the retina. Mutations in the myosin VIIA gene are associated with a variety of sensory neuronal disorders including the Usher syndrome 1B, characterized by deafness, vestibular dysfunction and blindness (retinitis pigmentosa), and two autosomal recessive hearing disorders, DFNA11 and DFNB2 in humans, and the shaker-1 phenotype in mice. Despite the importance of the myosin VIIA gene in the etiology of these syndromes, there is a lack of information on the molecular mechanism of this motor protein. The goal of this proposal is to characterize the motor properties of the myosin VIIA protein by determining if myosin VIIA is a processive motor and by studying the effect of the myosin VIIA mutations on the ATPase activity and motility. Truncated forms of the wild type and three shaker mutants of the myosin VIIA will be expressed in a baculovirus/insect expression system along with the calmodulin light chain. We will carry out steady-state experiments to determine the overall rate of the hydrolysis or the "output" of the motor, and transient kinetic measurements to determine selected rate constants for the substrate binding to and product dissociation from the wild type and mutant myosin VIIA and actomyosin VIIA complexes. Thus the rate limiting step of the myosin VIIA ATPase cycle and the duty ratio (the fraction of the ATPase cycle time the myosin spends tightly bound to actin) of the motor will be determined. The rate constants determined by these studies will aid us in designing the analytical ultracentrifugation and the in vitro motility studies. Analytical ultracentrifugation experiments will be conducted so that the molecular weight of the wild type and mutant myosin VIIA proteins can be accurately measured. These studies will also determine whether myosin VIIA forms dimers. The velocity of the wild type and mutant myosin VIIA will be determined by measuring the motility of the rhodamin labeled actin filaments sliding over the myosin VIIA molecules (in vitro motility). In the single molecule motility measurements, fluorescently labeled CaM light chain will be added to the myosin VIIA and the movement of the molecule will be measured by Total Internal Reflection Fluorescence microscopy (TIRF). The run length (the distance that the myosin VIIA molecule moves on the actin filament without dissociating from it), the step size (the distance between the two heads) thus the extent to which the different myosin VIIA constructs are processive will be determined. These studies will aid to elucidate the molecular mechanism of the myosin VIIA motor protein and increase our understanding of the myosin VIIA related deafness, and blindness. The biochemical, structural and motile properties of the wild type and mutant myosin VIIA proteins will be studied, to delineate the molecular mechanism of the myosin VIIA related deafness, vestibular dysfunction and blindness. [unreadable] [unreadable]